The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA|purification methods]] and there exists several protocols for [[PCR]]

The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA|purification methods]] and there exists several protocols for [[PCR]]

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===Restriction Digest===

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Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]].

===Ligation===

===Ligation===

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Just see [[DNA ligation]]

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The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]].

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====Reagents====

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'''Reagents'''

*[[T4 DNA ligase]]

*[[T4 DNA ligase]]

*10x T4 DNA Ligase Buffer

*10x T4 DNA Ligase Buffer

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*Purified, linearized insert (likely in H2O or EB)

*Purified, linearized insert (likely in H2O or EB)

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====Method====

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'''Method'''

#Mix reagents for a 10 μL reaction.

#Mix reagents for a 10 μL reaction.

#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C

#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C

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Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]].

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====Reagents====

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'''Reagents'''

*[[Competent cells]]: XL2-Blue (Stratagene 200150).

*[[Competent cells]]: XL2-Blue (Stratagene 200150).

**Learn how to make your own by [[Preparing_chemically_competent_cells]]

**Learn how to make your own by [[Preparing_chemically_competent_cells]]

Media and plates

Procedure

Enriching the insert

The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various purification methods and there exists several protocols for PCR

Restriction Digest

Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See Restriction digest.

Ligation

The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see DNA ligation.